Platelets are small, anuclear blood cells which are fairly quiescent under normal conditions but which respond immediately to vascular injury by adhesion, activation, aggregation, and thrombus formation. The primary function of platelets is to stop blood loss after tissue trauma and exposure of the subendothelial matrix. It is well known that damage to a blood vessel can expose extracellular matrix components to the blood, particularly von Willebrand factor (VWF), collagen, fibronectin, thrombospondin, and laminin. Interaction of platelets with these exposed molecules results in activation of the platelet cells.
While platelets have long been recognized as having a predominant role in hemostasis and thrombosis, it is becoming increasingly recognized that platelets may also play a significant role in a variety of other disorders such as inflammation, tumor growth and metastasis, and immunological host defense. Accordingly, platelet receptor proteins are attractive targets for regulation of platelet function as a means of treating or preventing platelet-mediated disorders.
Glycoprotein VI (GPVI) is a particularly attractive target as it is a transmembrane protein specifically expressed on platelets. Cell-specific molecules having extracellular exposure offer both accessibility to a therapeutic moiety and the potential for minimal—if any—adverse side effects, due to the limited expression profile.
Glycoprotein VI (GPVI) is a major platelet signaling receptor for collagen shown to have a role in generating intracellular signals that mediate platelet activation (Watson et al. Platelets, 2000, 11:252-258). GPVI is a 62-65 kDa glycoprotein in the Ig superfamily consisting of two Ig C2 loops that contain a collagen binding domain, representing potential drug target sites. The protein is very highly glycosylated and sialylated in vivo, and a single glycosylation site is found on the outer Ig-C2-like domain. As a member of the immunoglobulin superfamily, GPVI is related to natural killer receptors. Its signaling may be mediated indirectly through the γ-chain of FcR or directly through the GPVI cytoplasmic domain. The FcR γ-chain contains an immunoreceptor tyrosine-based activation motif and in conjunction with the non-tyrosine kinase SYK and the adapters LAT and LCP2 leads to the activation of phospholipase C2 and associated intracellular signaling pathways (Lankhof et al. Thromb Haemost, 1996, 75:950-958).
The importance of GPVI in the physiology and clinical events associated with clot formation has been recognized and is supported by epidemiologic associations of GPVI levels with onset of acute coronary syndrome (ACS) and stroke events (Bigalke et al. American Heart Journal, 2008, 156:193-200; Bigalke et al., European Journal of Neurology, 2009, 101:911-915), and the resistance to thrombosis demonstrated in GPVI deficient mice (Denis et al. Arteriosclerosis, Thrombosis and Vascular Biology, 2007, 27:728-739). Further, Gawaz et al have shown that radiolabeled GPVI could be used in scintillographic imaging of vascular lesions in mice as it binds specifically to the injured region, indicating that collagen is exposed at these sites (Thromb Haemost., 2005, 93:910-913). Penz et al. (FASEB J. 2005, 19:898-909) have shown that human atheromatous plaques from patients with carotid stenosis contained collagen type I and type III structures that were able to activate platelets. Further, blockage or absence of GPVI was able to prevent thrombus formation, whereas blockage of α2β1 had little effect. Similarly blocking collagen with anti-collagen antibodies or degrading it with collagenase prevented thrombus formation.
More recently, GPVI has been linked to a broader role in disorders associated with platelet malfunctioning and abnormal collagen expression. This is due in part to the fact that platelets function both in adhesion following vascular injury as in the case of thrombosis, and in the release of a variety of inflammatory mediators and cytokines. Moreover, following reaction of platelets with a site of vascular injury, subsequent activation of the platelets results in release of cytokines and other regulatory molecules. Thus, although it may seem that the disorders associated with abnormal activation of platelets are diverse, these disorders are linked by their dependence on platelet function. Accordingly, inhibiting or preventing activation of platelets can provide valuable therapeutic effects.
Platelet-mediated disorders include vascular diseases as well as a variety of disorders associated with high-risk diabetes. Inflammatory disorders shown to be platelet-mediated include inflammatory arthritides and scleroderma. The role of inflammation and white blood cell activity has long been known in inflammatory joint disease. More recently, the presence of platelets in synovial fluid of inflamed joints has been identified (Boilard et al., Science, 2010, 327:580-583). Moreover, platelet microparticles in joint fluid from patients suffering from inflammatory arthritis have been shown to be proinflammatory, eliciting cytokine responses from synovial fibroblasts via IL-1. Both pharmacologic and genetic approaches showed that GPVI plays a key role in this proinflammatory nature of platelets in arthritis.
Other disorders shown to be associated with GPVI expression on the surface of platelets include experimental tumor metastasis (Jain et al., J. Thromb Haemostasis, 2009, 7:1713-1717), diabetes (Cabeza et al., 2004, 53:2117-2121) and infection by Hepatitis C virus (Zahn et al., Diabetes, 2004, 53:2117-2121).
Despite the expanding role GPVI has been shown to have in platelet function and related physiological maladies, efforts to discover and develop antagonists of this receptor have been limited. Active control, or modulation, of the intensity and duration of antiplatelet therapy can provide significant clinical benefit. Therefore, there remains a need in the art for modulatable agents designed to specifically target and regulate the function of the GPVI protein.